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The potential of the A549 type II epithelial cell line to release granulocyte colony-stimulating factor (G-CSF) as neutrophil chemotactic activity (NCA) was evaluated. It was demonstrated that A549 cells released G-CSF as NCA in response to interleukin-1 beta, tumor necrosis factor-alpha, lipopolysaccharide and bradykinin. The expression of G-CSF mRNA was augmented in response to each stimulus. These suggested that alveolar type II cells may produce G-CSF as NCA and may participate in the regulation of neutrophil extravasation into the lung.

Experiments were performed to investigate the capacity of the human lung fibroblast (HLF) to induce monocyte chemokinetic activity (MCA) through the release of the transforming growth factor-beta, leukotriene B4 (LTB4) and the granulocyte-macrophage colony-stimulating factor. The attenuation of the MCA and its lowest molecular weight peak was attributed to an LTB4-receptor antagonist. These findings emphasize the role of HLFs as modulators of monocyte recruitment in the lungs.

A study was conducted to investigate the influence of acetylcholine (ACh) on alveolar macrophages (AM) in releasing inflammatory cell chemotactic activities. A Ficoll-Hypaque density centrifugation was utilized to separate red blood cells and neutrophils from mononuclear cells. Results showed that ACh plays a critical role in inflammatory cell recruitment and stimulates the release of lipoxygenase-based chemotactic activities.

The role of type II pneumocytes in the maintenance of host defense and alveolar immune environment was analyzed in A549 cell lines. Analysis of A549 cell supernatant fluids indicated the ability of type II pneumocytes to generate chemotactic factors that promote chemoattractant activity for monocytes in a time-dependent manner. Furthermore, the A549 supernatant contained transforming growth factor-beta, monocyte chemoattractant protein-1 and leukotriene B4.

Cyclophosphamide is an alkylating antineoplastic agent used in several conditions. However, little is known about the mechanism of its pulmonary toxicity. In the present study, we determined that human lung fibroblasts release activity for neutrophils and monocytes in response to cyclophosphamide in a dose- and time-dependent manner. Checkerboard analysis revealed that both neutrophil and monocyte activities were chemotactic. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular-sieve column chromatography revealed that both neutrophil (NCA) and monocyte (MCA) chemotactic activities had multiple peaks. NCA was inhibited by a leukotriene [B.sub.4] receptor antagonist and anti-interleukin-8 and anti-granulocyte colony-stimulating factor antibodies. MCA was attenuated by a leukotriene [B.sub.4] receptor antagonist and anti-monocyte chemoattractant protein-1 and anti-granulocyte-macrophage colony-stimulating factor antibodies. The concentrations of interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor significantly increased in response to cyclophosphamide. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to cyclophosphamide. interstitial lung disease; interleukin-8; granulocyte colony-stimulating factor; monocyte chemoattractant protein-1; granulocyte-macrophage colony-stimulating factor

Koyama, Sekiya, Etsuro Sato, Hiroshi Nomura, Keishi Kubo, Masakazu Miura, Tetsuji Yamashita, Sonoko Nagai, and Takateru Izumi. The potential of various lipopolysaccharides to release IL-8 and G-CSF. Am J Physiol Lung Cell Mol Physiol 278: L-658-L666, 2000.--Lipopolysaccharide (LPS) derived from Pseudomonas aeruginosa is less cytotoxic than that from Escherichia coli. But P. aeruginosa induces a prominent sustained lung inflammation as in cystic fibrosis and diffuse panbronchiolotis. The present study examined the potential for several LPSs obtained from E. coli and P. aeruginosa to release neutrophil chemotactic activity (NCA) from lung cells. LPSs differently stimulated A549 cells, BEAS-2B cells, and lung fibroblasts to release NCA [P. aeruginosa [is greater than] E. coli 0127:B8 (Difco) [is greater than] E. coli 055:B5 (Sigma) [is greater than] E. coli 026:B6 (Sigma)]. E. coli 0127;B8 (Sigma) and 0111:B4 (Sigma) did not stimulate these cells. NCA was chemotactic by checkerboard analysis. Molecular-sieve column chromatography revealed three chemotactic peaks. The release of NCA was inhibited by cycloheximide and lipoxygenase inhibitors. Experiments with blocking antibodies suggested that much of the NCA was secondary to the release of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF). Thus we examined the concentrations of IL-8 and G-CSF and found that the potency of the various LPSs to stimulate NCA closely paralleled the potency in releasing A549 cells was observed. Finally, the release of IL-6 showed similar results. These data suggest that P. aeruginosa LPS may stimulate lung cells to release more NCA than E. coli LPSs, leading to sustained lung inflammation. Pseudomonas aeruginosa; interleukin-8; granulocyte colony-stimulating factor; interleukin-6; A549 cells; BEAS-2B cells; fibroblasts.

Sato, Etsuro, Sekiya Koyama, Akemi Takamizawa, Takeshi Masubuchi, Keishi Kubo, Richard A. Robbins, Sonoko Nagai, and Takateru Izumi. Smoke extract stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activities. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1149-L1157, 1999.--Accumulation of monocytes and neutrophils and fibrous distortion of the airway are characteristics of airway disease secondary to smoking. The presence of inflammatory cells and fibrosis correlate, and, therefore, we postulated that lung fibroblasts might release chemotactic activity for neutrophils and monocytes in response to smoke extract. To test this hypothesis, human fetal lung (HFL1) fibroblasts were cultured, and the supernatant fluid was evaluated for neutrophil (NCA) and monocyte (MCA) chemotactic activities with a blind well chamber technique. HFL1 fibroblasts released chemotactic activity in response to smoke extract in a dose- and time-dependent manner (P [is less than] 0.05). Checkerboard analysis showed that the activity was predominantly chemotactic. Partial characterization of the released chemotactic activity revealed that the activity was partly heat labile, trypsin sensitive, and ethyl acetate extractable. Lipoxygenase inhibitors and cycloheximide inhibited the release of both NCA and MCA. Molecular-sieve chromatography revealed that NCA and MCA were heterogeneous. NCA was inhibited by anti-human interleukin (IL)-8 and anti-granulocyte colony-stimulating factor antibodies and a leukotriene (LT) [B.sub.4]-receptor antagonist. Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) and anti-monocyte chemoattractant protein (MCP)-1 antibodies and an [LTB.sub.4]-receptor antagonist inhibited MCA. Immunoreactive IL-8, granulocyte colony-stimulating factor, GM-CSF, and MCP-1 significantly increased in culture supernatant fluid in response to smoke extract. Finally, smoke extract augmented the expression of mRNAs of IL-8, GM-CSF, and MCP-1. These data demonstrate that lung fibroblasts release NCA and MCA in response to smoke extract and suggest that lung fibroblasts may modulate the inflammatory cell recruitment into the lung. interleukin-8; granulocyte colony-stimulating factor; monocyte chemoattractant protein-1; granulocyte-macrophage colony-stimulating factor; fibrosis; chemotaxis; smoking

Sato, Etsuro, Keith L. Simpson, Matthew B. Grisham, Sekiya Koyama, and Richard A. Robbins. Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L543-L549, 1999.--Peroxynitrite, an oxidant generated by the interaction between superoxide and nitric oxide (NO), can nitrate tyrosine residues, resulting in compromised protein function. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that attracts, monocytes and has a tyrosine residue critical for function. We hypothesized that peroxynitrite would alter MCP-1 activity. Peroxynitrite attenuated MCP-1-induced monocyte chemotactic activity (MCA) in a dose-dependent manner (P [is less than] 0.05) but did not attenuate leukotriene [B.sub.4] or complement-activated serum MCA. The reducing agents dithionite, deferoxamine, and dithiothreitol reversed the MCA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, an NO donor, or superoxide generated by xanthine and xanthine oxidase did not show an inhibitory effect on MCA induced by MCP-1. The peroxynitrite generator 3-morpholinosydnonimine caused a concentration-dependent inhibition of MCA by MCP-1. Peroxynitrite reduced MCP-1 binding to monocytes and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite, with subsequent inhibition of MCP-1 binding to monocytes, and suggest that peroxynitrite may play a role in regulation of MCP-1-induced monocyte chemotaxis. nitric oxide; monocyte migration; superoxide; nitrotyrosine; monocyte chemoattractant protein-1

A study has been conducted to investigate the side effects of the use bleomycin on cancer patients. To determine these effects, tests were done on harvested A549 cell supernatant fluid were evaluated for neutrophil and monocyte chemotactic activities. Findings have confirmed that bleomycin stimulates type II epithelial cells to release chemotactic activities and promote cell inflammation in the lungs, which could develop into diffuse interstitial lung fibrosis.

Eotaxin, a potent eosinophil-specific chemotactic factor, is increased in the lower respiratory tract of asthma patients. Recently, lung fibroblasts have been reported to produce eotaxin and their activation can be modulated by inflammatory cytokines. To test the hypothesis that inflammatory cytokines modulate the eotaxin release from lung fibroblasts, we investigated the potential of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ) to induce the release of eotaxin and eotaxin mRNA by the human fetal lung fibroblast cell line, HFL-1, was evaluated. HFL-1 released eotaxin constitutively without stimulation, but IL-1β or TNF-α stimulated eotaxin release in a dose- and time-dependent manner. IL-1β or TNF-α treatment of HFL-1 also resulted in the augmented expression of eotaxin mRNA. Although IFN-γ alone had negligible effect on eotaxin release and mRNA expression, IFN-γ induced a significant, concentration-dependent attenuation of eotaxin release and eotaxin mRNA expression from HFL1 stimulated with IL-1β or TNF-α . These findings are consistent with the concept that lung fibroblast-derived eotaxin may in part be responsible for the eosinophil infiltration observed in the airways ofasthmatic patients and that network ofcytokines may modulate the eosinophil recruitment to the airways by stimulation of fibroblasts to release eotaxin. [ABSTRACT FROM AUTHOR]